Outcomes of mitochondrial long chain fatty acid oxidation and carnitine defects from a single center metabolic genetics clinic

Background Mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects are a group of inherited metabolic diseases. We performed a retrospective cohort study to report on the phenotypic and genotypic spectrum of mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects as well as their treatment outcomes. Methods All patients with mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects were included. We divided patients into two groups to compare outcomes of those treated symptomatically (SymX) and asymptomatically (AsymX). We reviewed patient charts for clinical features, biochemical investigations, molecular genetic investigations, cardiac assessments, neuroimaging, treatments, and outcomes. Results There were 38 patients including VLCAD (n = 5), LCHAD (n = 4), CACT (n = 3), MAD (n = 1), CPT-I (n = 13), CPT-II (n = 3) deficiencies and CTD (n = 9). Fourteen patients were diagnosed symptomatically (SymX), and 24 patients were diagnosed asymptomatically (AsymX). Twenty-eight variants in seven genes were identified in 36 patients (pathogenic/likely pathogenic n = 25; variant of unknown significance n = 3). Four of those variants were novel. All patients with LCHAD deficiency had the common variant (p.Glu474Gln) in HADHA and their phenotype was similar to the patients reported in the literature for this genotype. Only one patient with VLCAD deficiency had the common p.Val283Ala in ACADVL. The different genotypes in the SymX and AsymX groups for VLCAD deficiency presented with similar phenotypes. Eight patients were treated with carnitine supplementation [CTD (n = 6), CPT-II (n = 1), and MAD (n = 1) deficiencies]. Thirteen patients were treated with a long-chain fat restricted diet and MCT supplementation. A statistically significant association was found between rhabdomyolysis, and hypoglycemia in the SymX group compared to the AsymX group. A higher number of hospital admissions, longer duration of hospital admissions and higher CK levels were observed in the SymX group, even though the symptomatic group was only 37% of the study cohort. Conclusion Seven different mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects were present in our study cohort. In our clinic, the prevalence of mitochondrial long-chain fatty acid oxidation and carnitine defects was 4.75%. Supplementary Information The online version contains supplementary material available at 10.1186/s13023-022-02512-5.

The characteristic clinical presentations include acute hypoketotic hypoglycemia, encephalopathy, cardiomyopathy and myopathy [13,14]. The symptoms can be precipitated by infections or prolonged fasting which results in hypoglycemia, elevated creatine kinase (CK), liver enzymes, lactate and ammonia levels. Plasma acylcarnitine profile and total and free carnitine levels can help identify specific enzyme or transporter deficiencies. The confirmation is by molecular genetic testing of candidate genes. Targeted next generation sequencing panel for myopathy and rhabdomyolysis typically includes these disorders [1,15].
To report outcomes of mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects, we performed a retrospective cohort study in the metabolic genetic clinic at our institution. We report on the phenotypic and genotypic spectrum of mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects as well as their treatment outcomes. Furthermore, we compare the outcomes of patients identified by the manifestation of symptoms (SymX group) and asymptomatically by positive newborn screening or positive family history (AsymX group) in our study.

Methods
The Research Ethics Office, Health Research Ethics Board, University of Alberta (Study ID: Pro00108842) approved this retrospective cohort study. All patients with the mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects were included. We divided patients into two groups to compare outcomes of those treated symptomatically and asymptomatically including Group 1) SymX group: diagnosed after the onset of symptoms; Group 2) AsymX group: diagnosed by positive family history or positive newborn screening (NBS).
We reviewed patient charts for clinical features, biochemical investigations, molecular genetic investigations, cardiac assessments, neuroimaging, treatments, and outcomes. We entered all information into an Excel database (Microsoft Corp., Redmond, WA, U.S.A.).
Molecular genetic investigations using patient and parent DNA samples were performed in clinical molecular genetic laboratories according to their methods. We applied American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) variant classification guidelines for interpretation of variants [22]. We also searched all variants in the Genome Aggregation Database (gnomAD) (http:// gnomad. broad insti tute. org/ about) for their allele frequency in the general population [23].
We performed statistical analysis using R statistical software (version 4.0.2). Results are given as mean ± SD (range). Non-parametric Fisher's exact and Wilcoxon rank-sum tests were chosen to compare outcomes between groups as indicated where appropriate. Results were considered statistically significant with a p-value of < 0.05.

Results
There were 38 patients (16 males, 22 females) from 27 unrelated families diagnosed with mitochondrial longchain fatty acid oxidation and carnitine metabolism defects. Their current average age was 15.3 ± 16.9 standard deviation (SD) years (range 3 months-55 years). There were 15 adults (> 18 years of age) (average age of 36.5 ± 11.2 SD years and age range of 22-55 years) and Keywords: Mitochondrial long-chain fatty acid oxidation, Carnitine metabolism defects, Medium chain triglycerides, Long-chain fat restricted diet, Newborn screening 23 children (average age of 4.5 ± 3.1 SD years and range of 3 months-12 years). The mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects diagnosed in our patient cohort were VLCAD (n = 5), LCHAD (n = 4), CACT (n = 3), MAD (n = 1), CPT-I (n = 13), CPT-II (n = 3) deficiencies and CTD (n = 9). The CTD, LCHAD and VLCAD deficiencies are included in the newborn screening programs in our Province of Alberta. Fourteen patients were diagnosed symptomatically and were included in the SymX group. Their average age was 21.4 ± 18.3 SD years (range 7 months-55 years). Twentyfour patients were included in the AsymX group and were diagnosed either by positive NBS (n = 9) (average age of 5.8 ± 3.4 SD years and range of 3 months-12 years) or by positive family history (n = 15) (average age of 21 ± 19.1 SD years and range of 5 months-53 years). We summarized the type of mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects, clinical features, biochemical features, genotypes, cardiac investigations, and treatments of patients in the SymX group in Table 1 and in the AsymX group in Table 2. Additionally, NBS results of the AsymX group are listed in Additional file1: Table S1.
Thirty-six patients had molecular genetic confirmation of their specific mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects. All variants identified in our study cohort were re-classified using ACMG/AMP criteria (Additional file 1: Table S2). Unfortunately, two patients (CPT-II n = 1; CTD n = 1) did not have molecular genetic investigations due to the limited publicly available funding for molecular genetic investigations at the time of their diagnosis. In one patient with LCHAD deficiency, a single variant was identified. There were 28 different variants from 36 patients including 25 pathogenic or likely pathogenic variants and three variants of unknown significance. The most common variant type was missense variant (n = 20). Fourteen patients had compound heterozygous variants and 21 patients had homozygous variants (Tables 1 and 2). Four of the variants were not reported in the literature previously including CPT2 (n = 2), and SLC25A20 (n = 2).
Eight patients were treated with carnitine supplementation [CTD (n = 6), CPT-II (n = 1), and MAD (n = 1) deficiencies]. The average carnitine dose was 48.6 mg/ kg/day (range 6.1-125 mg/kg/day). Thirteen patients were treated with a long-chain fat restricted diet and MCT supplementation (Table 3). Diet therapy recommendations were individualized based on the type of mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects as well as clinical judgement regarding disease severity. The most severe patients were prescribed the greatest long chain fat restrictions and shortest fasting allowances. Recommendations for long chain fat restriction ranged from 5% of total energy for severe phenotypes to 35% of total energy for mild or moderate phenotypes. MCT was supplemented as an alternative energy source to meet total fat requirements, providing 0 to 35% of total energy. MCT supplementation was provided using either medical formulas (Lipistart) or module forms (MCT oil, MCT procal, Betaquick, or Liquigen). Age dependent maximum hours of fasting recommendations were applied. The longest allowed overnight fasting was up to 12 h in older children and adults. For illness management, carbohydrate was provided either through glucose polymers (such as Solcarb) or IV glucose, depending on the patient's ability to tolerate feeding (and thus stay at home) versus require admission. MCT supplementation was also used in some cases as an alternative energy source to long-chain fat. Details of complex carbohydrate and/or MCT intakes prior to exercise as well as the compliance and moderation in exercise level are summarized in Additional file 1: Table S3.

Clinical and biochemical features and treatment outcomes of patients in the SymX group (n = 14)
There were 14 patients with CPT-I (n = 5), CPT-II (n = 3), LCHAD (n = 1), VLCAD (n = 2), CACT (n = 2) and MAD (n = 1) deficiencies. The average current age was 21.4 ± 18.3 SD years (range 7 months-55 years). The average age of symptom onset was 4.5 ± 7.4 SD years (range newborn-23 years). The average age of diagnosis was 10.6 ± 16.1 SD years (range newborn-47 years). Two patients with CPT-I deficiency presented at birth with hypoglycemia and two patients with CACT deficiency presented with symptoms prior to receiving NBS results. The average lack of time between the symptom onset and diagnosis was 6.1 ± 12.1 SD years (range newborn-43 years). The average duration of follow-up was 10.6 ± 8.8 years SD (range 7 months-24.2 years). The symptom frequency for each disorder is summarized in Table 4.
Seven patients [(CPT-II (n = 3), LCHAD (n = 1), VLCAD (n = 2), MADD (n = 1) deficiencies] were treated with a long-chain fat restricted diet. The average age of diet initiation was 20.4 ± 18.2 SD years (range newborn-48 years). The average recommended long-chain fat intake was 19% ± 0.10 SD of total daily intake (range 5-35%), whereas the average of actual long-chain fat intake was 24% ± 0.1 SD of total daily intake (range 5-44%). The average recommended MCT intake was 16% ± 0.1 SD of daily total intake (range 0-35%), whereas the average actual MCT intake was 13% ± 0.1 SD of total daily intake (range 0-21%). We recommended pre-exercise complex carbohydrates in two patients (CPT-II n = 1; and MAD n = 1 deficiencies) to prevent rhabdomyolysis associated with exercise. Additionally, we recommended pre-exercise complex carbohydrates and MCT oil (in any form) in four patients (VLCAD n = 1; LCHAD n = 1; and CPT-II n = 2 deficiencies) to prevent rhabdomyolysis associated with exercise (Additional file 1: Table S3). Three patients were compliant with these recommendations including patients with CPT-II (n = 1), VLCAD (n = 1) and MAD (n = 1) deficiencies. Three patients were not compliant with our recommendations and for two of them with CPT-II deficiency we recommended limited level of exercise to prevent repeated rhabdomyolysis episodes. One non-compliant patient with LCHAD deficiency did not exercise. None of the patients received additional oil or cornstarch supplementation at the last assessment. One patient with CPT-II deficiency was noncompliant since the diagnosis (at age 9 years) due to very high exercise levels and failure to consume an appropriate amount of carbohydrates leading to several hospital admissions for the treatment of rhabdomyolysis. One patient with LCHAD deficiency had difficulty in being compliant with the long-chain fat restricted diet leading to multiple hospital admissions for rhabdomyolysis since the diagnosis. The adherence to the diet was improved with the use of nasogastric feeds, but this was not sustainable for long-term.
Adherence to diet recommendations was suboptimal in the SymX group. 50% (7 out of 14) of the patients were prescribed diet therapy including long chain fat restriction and, in some cases, MCT supplementation. 43% (3 out of 7) of the patients met their long chain fat goals within 1% of max recommendations or took less long chain fat than prescribed. Those that did not meet their long chain fat goals were not monitoring daily fat intake so had day-to-day variability in fat consumption. 71% (5 out of 7) of the patients were prescribed MCT supplements, where 60% (3 out of 5) of the patients met their goals. Two patients, who did not meet MCT goals, struggled with gastrointestinal side effects. One patient experienced significant abdominal discomfort and bloating. Another patient's baseline irritable bowel syndrome like symptoms (abdominal discomfort and diarrhea) were exacerbated.
The patient with LCHAD deficiency had 16 acute intercurrent illnesses. One patient with VLCAD deficiency had four acute intercurrent illnesses and the other patient with VLCAD deficiency had 7 acute intercurrent illnesses. One patient with CACT deficiency reported one acute intercurrent illness treated at home. The number of episodes of elevated CK levels ranged from 0 to 19. The average peak CK during acute intercurrent illness or catabolism was 72,783.7 ± 60,708.7 SD U/L (range 246-197,836) (Additional file 1: Table S3). We had details for hospital admission and the CK levels for six patients as depicted in Fig. 1.

Clinical and biochemical features and treatment outcomes of patients in the AsymX group (n = 24)
Twenty-four patients were asymptomatic at the time of diagnosis and were identified by positive NBS (n = 9) or by positive family history (n = 15) ( Table 2). Patients with positive NBS included VLCAD (n = 2), LCHAD (n = 1), CPT-I (n = 1) deficiencies and CTD (n = 5). Patients with positive family history included CPT-I (n = 7), LCHAD (n = 2), VLCAD (n = 1), and CACT (n = 1) deficiencies and CTD (n = 4). The average current age was 15.3 ± 16.9 SD years (range 3 months-53 years). The average duration of follow-up was 5.6 ± 3.8 SD years (range 3 months-12 years) in 19 patients, as five asymptomatic patients with CPT-I deficiency were only seen once.
Nine patients developed symptoms later including three patients with positive family history and six patients identified by positive NBS. The average lack of time between the diagnosis and symptom onset was 3.1 ± 2.8 SD years (range newborn-8 years). In three patients with positive family history, the symptom onset was during intercurrent illness including two patients with LCHAD deficiency and one patient with CACT deficiency. They presented with rhabdomyolysis, lethargy, hypotonia and hypoglycemia. The average age of symptom onset was 2 ± 3.5 years SD (range newborn-6 years). The average time between the diagnosis and symptom onset was 7.9 ± 13.8 months SD (range newborn-2 years). Six patients identified by positive NBS had subsequent symptom onset including retinopathy in one patient with LCHAD deficiency, rhabdomyolysis in two patients with LCHAD and myalgia in two patients with VLCAD deficiency ( Table 2). The average age of symptom onset was 3.2 ± 2.7 SD years (range 1 month-8 years).
All patients with CPT-I deficiency (n = 8) were asymptomatic. One of the patients with CPT-I deficiency had atrial septal aneurysm with a residual patent foramen ovale and atrial septal defect, residual patent ductus arteriosus, and mild mitral incompetence in echocardiography at age one month. None of the patients with CTD, CACT, LCHAD and VLCAD deficiencies had cardiomyopathy.
All patients received illness and emergency management. None of the patients with CPT-I deficiency required any treatments. Eight patients with CTD were treated with carnitine supplementation; one was not compliant with carnitine supplementation. All patients with LCHAD (n = 3) and VLCAD (n = 3) deficiencies received long-chain fat restricted diet. The average age of diet initiation was 2.2 ± 4.8 SD months (range newborn-13 months). The average recommended intake of long-chain fat was 18% of total energy intake ± 0.1 SD (range 10-25%). Whereas the average actual intake of long-chain fat was 13% ± 0.1 SD of total energy intake (range 7-26%). The average recommended intake of MCT was 20% of total energy intake ± 0.1 SD (range 13-35%). Whereas the average actual intake of MCT was 19% of total energy intake ± 0.1 SD (range 13-35%). Four patients received walnut oil supplementation (average 1.63 ml, range 0.5-4.0 ml), one patient received flax oil supplementation (0.5 ml), one patient received canola oil supplementation (30 ml), and two patients received cornstarch supplementation (average 0.8 mg/kg/day, range 0.6-0.9 mg/kg/day). The ASympX group demonstrated good adherence to dietary recommendations. 25% (6 out of 24) of the patients were prescribed diet therapy including long chain fat restriction and MCT supplementation. 100% of the patients met their long chain fat goals within 1% of max recommendations or took less long chain fat than prescribed. 83% (5 out of 6) of the patients met their MCT goals. The exception was one child who refused MCT supplementation due to poor palatability. We recommended pre-exercise complex carbohydrates in two patients with VLCAD deficiency to prevent rhabdomyolysis associated with the exercise. Additionally, we recommended pre-exercise complex carbohydrates and MCT oil (in any form) in two patients (VLCAD n = 1; and LCHAD n = 1 deficiencies) to prevent rhabdomyolysis associated with the exercise (Additional file 1: Table 3). Three patients were compliant with these recommendations including patients with VLCAD (n = 2) and LCHAD (n = 1) deficiencies and none of these patients had a reduced exercise level. Unfortunately, we are not certain about the compliance of one patient with VLCAD deficiency as the patient is no longer followed by our clinic due to moving out of province. The average number of acute intercurrent illnesses was 6.3 ± 3.3 SD (range 2-10) in patients with LCHAD deficiency. The average number of acute intercurrent illnesses was 4.7 ± 3.8 SD (range 2-9) in patients with VLCAD deficiency. The patient with CACT deficiency reported two acute intercurrent illnesses treated at home. The number of acute episodes of elevated creatine kinase (CK) levels ranged from 0 to 10. The average peak CK during acute intercurrent illness or catabolism was 22,842.5 ± 32,740.3 U/L SD (range 386-70,000) (Additional file 1: Table 3). Details for hospital admission and the CK levels were available for four patients and depicted in Fig. 2.

Comparison of patients in the SymX group and AsymX group
The symptom frequency for each group is summarized in Table 4. We excluded three patients who were diagnosed due to their positive family history but reported symptoms when they were asked for more information ( Table 2). Statistically significant association was found for rhabdomyolysis, and hypoglycemia in the SymX group compared to AsymX group. The total number of hospital admissions was 16 in one patient with LCHAD deficiency in the SymX group (n = 1), whereas the total number of hospital admissions was 12 in three LCHAD patients in the AsymX group (n = 3) (Individual admissions: 6, 4, 2) (p = 0.500, Wilcoxon rank-sum). The total number of hospital admissions was seven in two patients with VLCAD deficiency in the SymX group (n = 2) (individual admissions: 3, 4), whereas the total number of hospital admissions were five in three VLCAD patients in the AsymX group (n = 3) (individual admissions: 1, 1, 3) (p = 0.224, Wilcoxon ranksum). The total number of hospital admissions was seven in five patients with CPT-I deficiency in the SymX group (n = 5) (individual admissions: 5, 0, 0, 1, 1), whereas none of the patients with CPT-I deficiency in the AsymX group (n = 8) (p = 0.0225, Wilcoxon rank-sum) had any hospital admissions. A higher number of hospital admissions, longer duration of hospital admissions and higher CK levels were observed in the SymX group, even though the SymX group was only 37% of the study cohort.

Discussion
We report 38 patients with seven different mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects in our retrospective study from a single metabolic genetics center at our institution. The two most common defects were CPT-I deficiency (27%) and CTD (24%). Interestingly, 38% of the patients with CPT-I deficiency were identified symptomatically, whereas none of the patients with CTD were diagnosed symptomatically but identified due to positive NBS of CTD in their children. Asymptomatic diagnosis and application of treatments appears to decrease the number and duration of hospital admissions as well as peak CK levels. Our study reports the genetic landscape of mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects in pediatric and adult patients from a single center. The prevalence of maternal CTD was about 1 in 33,000 in a study from Taiwan [24]. In 91 families with abnormal NBS for CTD, 14 mothers were identified with two variants in SLC22A5. Eleven of those mothers were asymptomatic and their age ranged from 21 to 43 years at the time of the publication. Fatigability was reported in three of those mothers (21%) [25]. Interestingly, in our study 33% of the mothers reported symptoms at the time of their identification due to the positive NBS of CTD in their children. It is possible that more mothers with maternal CTD may have symptoms and their diagnosis may improve their quality of life and prevent deaths secondary to cardiomyopathy.
The prevalence of CPT-I deficiency in live newborns in three Canadian Territories were reported in 2010. The study genotyped all newborns born in Yukon, Northwest Territories and Nunavut for the p.Pro479Leu variant in CPT1A in 2006. The homozygosity rate was 0%, 3%, and 64% respectively in those Canadian Territories [26]. The prevalence of hypoglycemia in term newborns from the Kivalliq region of Nunavut was 22% in newborns with homozygous p.Pro479Leu variant in CPT1A [27]. Interestingly, 80% of the SymX patients with CPT-I deficiency had hypoglycemia in our patient cohort. None of the individuals with AsymX CPT-I deficiency reported any symptoms after the genotypic diagnosis. It is important to genotype symptomatic individuals, as asymptomatic individuals do not report any symptoms to the best of our knowledge. If there is a positive family history for symptomatic CPT-I deficiency, newborns in that family can be genotyped to prevent symptoms by application of illness management and emergency letters.
Trauma exposure and post-traumatic stress disorder like behavior in mice were studied using plasma and cerebellum metabolomics analysis. Investigators identified that medium and long chain acylcarnitines were elevated including C8, C12, C14, C16, C18:1 and C18. Additionally, free carnitine was reduced [28]. Interestingly, five adult patients had either suspected or diagnosed mental health condition in our study. Depression was reported in one patient with LCHAD, one patient with CPT-II deficiencies and one patient with CTD. Depression and anxiety were reported in one patient with CPT-I deficiency. Attention deficit disorder was reported in one patient with MAD deficiency. It is important to monitor abnormalities in acylcarnitine and carnitine levels in these patients with mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects. This may increase our understanding of mental health conditions and help formulate better treatments.
Carnitine is a cofactor that transports long-chain fatty acids across the inner mitochondrial membrane for beta oxidation to provide energy to the skeletal and cardiac muscle. Energy demands of skeletal and cardiac muscle strongly require beta oxidation. Approximately 98% of the body's carnitine is located within cardiac and skeletal muscle tissues. Orally taken carnitine is transported to skeletal and cardiac muscle via carnitine transporters. The half-life of a single dose (2 g) of oral l-carnitine is 60.3 ± 15 min and requires frequent oral intake to maintain carnitine levels. Oral l-carnitine intake of 2 g twice a day increases muscle carnitine by 21% of baseline. Carnitine supplementation increases plasma carnitine levels in individuals with CTD but does not normalize completely. High dose carnitine supplementation (up to 200-250 mg/kg/day) is usually required in individuals with CTD. It has been shown that blood and urine carnitine levels are increased on l-carnitine supplementation. However, there was no measurable increase in intracellular carnitine concentrations. Plasma carnitine levels on oral l-carnitine supplementation do not reflect the tissue carnitine levels. Especially during physical activity, energy demands of skeletal and cardiac muscle increase. It has been shown that during exercise there is an impaired beta oxidation capacity in individuals with CTD which is partially restored by carnitine supplementation [29,30]. Unfortunately, high dose carnitine supplementation results in a fishy odor to the breath, sweat and urine. The frequent dosing and fishy body odor are major limiting factors for treatment compliance. There are unfortunately sudden deaths in childhood, adolescents, and adults with CTD. The sudden death is attributed to compliance problems, but it has not been proven to be correct. There are no extensive investigations to understand the mechanisms of sudden death in patients with CTD as well as how to prevent this devastating complication. It is timely to think about other treatments in addition to carnitine supplementation such as medium chain triglyceride supplementation or anaplerotic agent such as triheptanoin.
In nine patients with VLCAD deficiency, peripheral blood cells (including natural killer cells, B lymphocytes, T lymphocytes, dendritic cells, hematopoietic stem cells, and hematopoietic progenitor cells) were used to measure oxygen consumption rate and fatty acid oxidation rate. Patients with VLCAD deficiency showed impaired oxidation capacity and reduced total energy production. The supplementation of long-chain fatty acids resulted in the accumulation of long-chain fatty acid in CD8 + T cells, leading to impairment of mitochondrial functions, and decrease in the fatty acid metabolism in these cells [31]. It was previously reported that lymphocyte, monocyte, macrophage, and neutrophil functions were